The low temperature preservation of biological tissues and organs has been the subject of much research effort. Although organ banks similar to blood banks would have great medical utility, it has not been possible to successfully preserve clinically required whole organs or certain tissue sections by cryogenic methods. Organized tissues and organs, especially the heart and kidney, are particularly susceptible to mechanical damage from ice crystals formed during freezing. Efforts to protect tissues from damage during freezing have involved the use of chemicals known as cryoprotective agents which frequently become excessively concentrated during the freezing process and prove toxic to the biological material. In order to avoid damage caused by ice formation on freezing, methods have also been developed which employ solutes in amounts sufficient to greatly depress the freezing point of aqueous protective solutions, permitting the tissues or organs to be stored at low temperatures in a liquid state. Typical of such methods is the equilibrium method employed by Farrant (Nature, 205:1284-87, 1965) wherein the tissue or organ is incubated with a penetrating cryoprotectant such as dimethyl sulfoxide (DMSO) until the intra- and extra-cellular concentrations of DMSO are equilibrated. The concentration of DMSO is gradually increased and the temperature simultaneously gradually lowered without freezing until a sufficiently low temperature is obtained. Owing to the necessity of equilibrating DMSO across the cell membranes with restoration of isotonic volumes, while lowering the temperature, the process is very slow. Further, in order to sufficiently depress the freezing point of the preservation solution, very high concentrations of DMSO are necessary and must be introduced and removed at the subzero temperatures contemplated. Additionally, the same slow procedure must be employed in reverse on recovery (warming) of the tissue for use.
Accordingly, it is desirable to provide a method for the successful preservation of organs, tissues and other biological materials at very low temperatures which avoids the formation of ice crystals, minimizes the effective concentration of potentially harmful chemicals, and permits the rapid introduction and removal of cryoprotectants at feasible temperatures, without the necessity of elaborate equipment to monitor precise conditions of concentration and temperature. These advantages are obtained by the vitrification process of the present invention.
The principles of vitrification are well-known. Very generally, the lowest temperature a solution can possibly supercool to without freezing is the homogeneous nucleation temperaure T.sub.h, at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution. Vitrification solutions have a glass transition temperature T.sub.g, at which temperature the solution vitrifies, or becomes a non-crystalline solid, higher than T.sub.h. Owing to the kinetics of nucleation and crystal growth, it is effectively impossible for water molecules to align for crystal formation at temperatures much below T.sub.g.
On cooling most dilute aqueous solutions to the vitrification temperature (about -135.degree. C.), T.sub.h is encountered before T.sub.g, and ice nucleation occurs, which makes it impossible to vitrify the solution. In order to make such solutions useful in the preservation of biological materails by vitrification, it is therefore necessary to change the properties of the solution so that vitrification occurs instead of ice crystal nucleation and growth. While it is known that many solutes, such as commonly employed cryoprotectants like dimethyl sulfoxide (DMSO), raise T.sub.g and lower T.sub.h, solution concentrations of DMSO or similar solutes high enough to permit vitrification typically approach the eutectic concentration and are generally toxic to biological material. While it is also generally known that high hydrostatic pressures similarly raise T.sub.g and lower T.sub.h, vitrification of most dilute solutions by the application of pressure is either impossible or impractical. Further, for many solutions vitrifiable by the application of pressure, the required pressures cause unacceptably severe injury to unprotected biomaterials during vitrification thereof; for example, a pressure of only 1000 atm is lethal to unprotected kidney slices. These and other barriers to cryopreservation of biological materials have not been surmounted in the prior art.
A summary of the effects of increasing concentrations of solute at decreasing temperatures on the cryo-behavior of an exemplary solution at two (2) different pressures is presented in the graph in FIG. 1 (T.sub.m is the melting point or liquidus temperature of the solution).